?PROJECT SUMMARY: In vitro differentiation of cultured pluripotent stem cells (PSCs) provides a promising tool for the generation of specific cell types useful for cell replacement therapies. Despite rapid progress over the last decade, numerous challenges encompassing cell-production quantity, quality and safety remain to be addressed before full-fledged clinical application becomes a reality. Moreover, generation of functional organs from cultured PSCs, a much needed breakthrough to meet the rising demands for organ donors, remains improbable. This is largely due to our limited understanding of molecular and genetic processes underlying in vivo tissue/organ generation, which has been perfected through millions of years? evolution. Nature has evolved a sophisticated and robust system to generate functional tissues and organs during normal course of embryo development. The intrinsic genetic program works seamlessly with extrinsic developmental niches in a perfect spatiotemporal manner to enable embryonic cells to commit to specific cell lineages and be organized into higher-order tissue architectures. Blastocyst complementation is based on emptying these developmental niches created by knocking out gene(s) critical for the specific tissue/organ development and use donor chimeric-competent PSCs to colonize the vacant niche and generate desired tissues/organs of donor origin. With the booming of CRISPR/CAS9-mediated genome engineering gene knockout animals can be generated with high efficiency by co-injection of Cas9 mRNA and gRNAs directly into zygotes. Also, chimeric-competent primate PSCs have seen several promising developments very recently. By combining the strength of zygote genome editing and chimeric-competent primate PSCs, we propose to establish a novel interspecific blastocyst complementation system for the generation of functional organs in a large animal host, the pig. For this first study, we will focus on using PSCs derived from non-human primate species, to evaluate the efficacy and safety of our proposed strategy. Our study will provide critical information for future application into humans. If successful, this approach will lead to a paradigm shift in regenerative medicine and will help to overcome the shortage of organ donors.